Omicron XBB.1.16-Adapted Vaccine for COVID-19: Interim Immunogenicity and Safety Clinical Trial Results

(1) Background: The global coronavirus disease 2019 vaccination adapts to protect populations from emerging variants. This communication presents interim findings from the new Omicron XBB.1.16-adapted PHH-1V81 protein-based vaccine compared to an XBB.1.5-adapted mRNA vaccine against various acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains. (2) Methods: In a Phase IIb/III pivotal trial, adults previously vaccinated with a primary scheme and at least one booster dose of an EU-approved mRNA vaccine randomly received either the PHH-1V81 or BNT162b2 XBB.1.5 vaccine booster as a single dose. The primary efficacy endpoint assessed neutralization titers against the Omicron XBB.1.16 variant at day 14. Secondary endpoints evaluated neutralization titers and cellular immunity against different variants. Safety endpoints comprised solicited reactions up to day 7 post-vaccination and serious adverse events until the cut-off date of the interim analysis. Changes in humoral responses were assessed by pseudovirion-based or virus neutralization assays. (3) Results: At the cut-off date, immunogenicity assessments included 599 participants. Both boosters elicited neutralizing antibodies against XBB.1.16, XBB.1.5, and JN.1, with PHH-1V81 inducing a higher response for all variants. The PHH-1V8 booster triggers a superior neutralizing antibody response against XBB variants compared to the mRNA vaccine. A subgroup analysis consistently revealed higher neutralizing antibody responses with PHH-1V81 across age groups, SARS-CoV-2 infection history, and the number of prior vaccination shots. A safety analysis (n = 607) at the day 14 visit revealed favorable safety profiles without any serious vaccine-related adverse events. (4) Conclusions: PHH-1V81 demonstrates superiority on humoral immunogenicity compared to the mRNA vaccine against XBB variants and non-inferiority against JN.1 with a favorable safety profile and lower reactogenicity, confirming its potential as a vaccine candidate.

In response to Omicron XBB variants, global health authorities, including the World Health Organization Technical Advisory Group on COVID-19 Vaccine Composition, the European Centre for Disease Prevention and Control, and the European Medicines Agency, recommended updates to vaccine formulations [8,9].mRNA and protein-based vaccines targeting Omicron XBB have gained approval, addressing evolving variants effectively [9][10][11][12][13].The emergence of JN.1 highlights the ongoing pandemic challenges, emphasizing the critical need for vaccine advancement [14,15].
Further, recipients of PHH-1V have reported fewer adverse events (AEs) than mRNA vaccine recipients, with comparable breakthrough non-severe COVID-19 rates.As a recombinant protein-based vaccine, PHH-1V offers advantages such as high productivity, stability, and suitability for immunocompromised individuals [20].PHH-1V81, a newly adapted XBB vaccine, targets XBB.1.16receptor-binding domain (RBD) homodimer and was developed in response to the evolution of SARS-CoV-2.Like PHH-1V, PHH-1V81 is a recombinant protein-based vaccine but is tailored to enhance protection against the more recent Omicron variants [16].
The PHH-1V (BIMERVAX ® ; HIPRA) antigen is a heterodimer based on the fusion of two RBDs from SARS-CoV-2 spike protein, incorporating the RBDs from the B.1.351(Beta) and B.1.1.7 (Alpha) variants into a single peptide using recombinant deoxyribonucleic acid (DNA) technology.Expressed in a Chinese Hamster Ovary cell line, each 0.5 mL dose contains 40 µg of the active substance in phosphate-buffered saline with a squalene (SQBA) adjuvant [16].The PHH-1V81 antigen is a homodimer of two consecutive copies of the RDB from the Omicron XBB.1.16variant and is being developed as a booster dose, focusing on providing robust immunity against the prevalent Omicron sublineage [5,16].
Participants in the experimental arm received a single intramuscular dose of 40 µg of PHH-1V81, while subjects in the active comparator arm received a single intramuscular dose of 30 µg of Comirnaty Omicron XBB1.5 [21].
The primary efficacy endpoint is neutralization titers against the Omicron XBB.1.16variant at day 14.Secondary efficacy endpoints include neutralization titers against the Omicron XBB.1.16variant at days 91 and 182 and against the Wuhan, Omicron BA.1, Omicron XBB.1.5strains at days 14, 91, and 182.Safety endpoints comprise solicited systemic and local reactions up to day 7 post-vaccination, unsolicited AEs up to day 28 after vaccination, AEs of special interest (AESI) until study end, medically attended AEs, and serious AEs (SAE) throughout the study.
The interim analysis compares the immunogenicity and safety of PHH-1V81 (HIPRA) with BNT162b2 XBB.1.5(Pfizer [New York, NY, USA], BioNTech, Mainz, Germany) at baseline and day 14, including solicited AE at day 7 post-vaccination for participants who completed the day 14 visit, and SAE until the interim analysis cutoff date (12 December 2023).
In addition to planned immunogenicity assessments, a virus neutralization assay (VNA) was conducted in a random subset of serum samples to compare humoral immune response between vaccine arms against the Omicron JN.1 [25,26].

Participants
The trial enrolled adults aged 18 or older who provided informed consent, received a primary series of two doses and at least one booster dose of an EU-approved mRNA vaccine with last dose at least six months before inclusion, and tested negative for acute SARS-CoV-2 infection on day 0. Eligible participants had stable chronic diseases, while those with a previous SARS-CoV-2 infection must have been diagnosed at least 6 months before day 0 (Table S1).The trial, conducted at ten clinical sites in Spain, began enrollment on 15 November 2023 and, due to rapid recruitment, closed on 29 November 2023.

Randomization and Treatment Allocation
This double-blinded study ensured that participants, site staff (including those involved in drug preparation and administration), laboratory analysts, the sponsor, and the Clinical Research Organization (CRO) were unaware of the treatment allocations.Only unblinded hospital pharmacists or other qualified personnel prepared the booster doses, and unblinded staff members, who were not otherwise involved in the study procedures except for blood extraction, administered the treatments [18].Blinding was maintained until study finalization.A label system was used to disguise the syringe contents due to the visual distinction between the two vaccines.
Participants were randomly assigned to treatment arms in a 2:1 ratio to receive either a booster dose of PHH-1V81 (HIPRA XBB.1.16-adaptedvaccine, n = 408) or a booster dose of BNT162b2 Omicron XBB.1.5(Comirnaty ® Omicron XBB.1.5,Pfizer-BioNTech adapted vaccine, n = 204) using Interactive Response Technology (IRT).The allocation was stratified by age group, with approximately 90% of participants aged 18-64 years and 10% aged 65 years or older [18].An independent statistician, who is not involved in the study, generated the randomization scheme.Upon enrollment, each participant was assigned a randomization number exclusively for arm allocation.

Sample Size
In the HIPRA-HH-14 trial, sample size determination aimed to confirm PHH-1V81's non-inferiority to the comparator vaccine in inducing neutralizing antibody titers against Omicron XBB variants.For consistency with previous HIPRA-HH studies, the success criterion was defined as the upper bound of the two-sided 95% confidence interval (CI) around the GMT ratio BNT162b2 XBB.1.5/PHH-1V81,which should lie below 1.50.Superiority is demonstrated if the upper bound of the 95% CI of the GMT ratio BNT162b2: PHH-1V81 is below 1 [27].With a 2:1 randomization ratio, group sizes of 366 and 183 ensured 90% power at a one-sided 2.5% significance level.This resulted in 612 randomized subjects, with 408 in the PHH-1V81 arm.

Statistical Analysis
Descriptive analyses compared time points and treatment arms for each SARS-CoV-2 variant.Categorical variables were presented as cases and percentages, while continuous variables included non-missing observations, mean (or geometric mean), standard deviation (or geometric standard deviation), median, interquartile range, minimum, and maximum, without imputation for missing data.Efficacy analyses followed predefined hypotheses for non-inferiority, with the upper bound of the 95% CI determining claim validation.GMT and GMFR adjusted treatment for immunogenicity endpoints were estimated using Mixed Models for Repeated Measures (MMRM), while T-cell data were analyzed with mixed effects models.Values below the lower limit of quantification (LLOQ) were imputed as LLOQ, and PBNA values exceeding 20,480 were reanalyzed.

Ethical Considerations
The trial was conducted in accordance with national and international regulations [28][29][30].
The study protocol was approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) and by HM Hospitals Research Ethics Committee (23.10.2249-GHM).Trial registration numbers are NCT06181292; EU CT No: 2023-508458-25-00.

Results
Out of 913 screened, 905 subjects constituted the Intention-to-Treat (ITT) population (293 more than the calculated sample size).Among them, 603 were in the PHH-1V81 arm and 302 in the BNT162b2 XBB.1.5arm.Vaccine administration was confirmed for 800 subjects (536 in PHH-1V81; 264 in BNT162b2 XBB.1.5)as of 12 December 2023, forming the safety population for the interim analysis.Only 607 subjects (409 in PHH-1V81; 198 in BNT162b2 XBB.1.5)completed the day 14 visit with solicited AE information available for the interim analysis.Immunogenicity data at both baseline and day 14 visits were available for 599 participants (66.2% of ITT, 97.9% of target sample size), included in the modified Intention-to-Treat (mITT) population for immunogenicity assessments.Among them, 406 received the PHH-1V81, and 193 received the BNT162b2 XBB.1.5.No premature discontinuations occurred by the cutoff date (Figure 1).

Baseline Characteristics
Participants had a median age of 45 years (range: 18 to 88 years), with similar age distributions in both vaccine arms.Most subjects were female (59.3%), having received either three (66.9%)or four (33.0%) previous vaccination doses.Demographic characteristics were generally balanced between the vaccine arms (Table 1).

Figure 1.
The participants' disposition of HIPRA-HH14.The Enrolled Population (EP) is defined as all subjects who signed the Informed Consent Form.The Intention-to-treat Population (ITT) is defined as all subjects of the EP who are randomly assigned to treatment, regardless of the subject's treatment status in the study.The safety population (SP) set is defined as all randomized subjects who received the study drug.A total of *607 subjects (409 in PHH-1V81 arm and 198 in the BNT162b2 XBB.1.5arm) had completed the day 14 visit, and information on solicited adverse events was available at the cut-off date set on 12 December 2023.The modified ITT Population (mITT) is defined as all participants in the ITT who met the inclusion/exclusion criteria and received a dose of study drug, whose baseline and day 14 were available and who did not test positive for COVID-19 within 14 days of receiving the study drug.COVID-19: coronavirus disease.

Baseline Characteristics
Participants had a median age of 45 years (range: 18 to 88 years), with similar age distributions in both vaccine arms.Most subjects were female (59.3%), having received either three (66.9%)or four (33.0%) previous vaccination doses.Demographic characteristics were generally balanced between the vaccine arms (Table 1).

Figure 1.
The participants' disposition of HIPRA-HH14.The Enrolled Population (EP) is defined as all subjects who signed the Informed Consent Form.The Intention-to-treat Population (ITT) is defined as all subjects of the EP who are randomly assigned to treatment, regardless of the subject's treatment status in the study.The safety population (SP) set is defined as all randomized subjects who received the study drug.A total of *607 subjects (409 in PHH-1V81 arm and 198 in the BNT162b2 XBB.1.5arm) had completed the day 14 visit, and information on solicited adverse events was available at the cut-off date set on 12 December 2023.The modified ITT Population (mITT) is defined as all participants in the ITT who met the inclusion/exclusion criteria and received a dose of study drug, whose baseline and day 14 were available and who did not test positive for COVID-19 within 14 days of receiving the study drug.COVID-19: coronavirus disease.
Both vaccines significantly increased the number of antigen-specific IFN-γ + T-cells in response to in vitro PBMC re-stimulation with RBD peptide pools from the Omicron XBB.1.5,Omicron XBB.1.16,and Omicron JN.1 variants at day 14 post-booster compared to baseline.No significant differences were observed in IFN-γ + spot forming cells between the vaccine arms (Figure 7 and Table S7).
An SAE is defined by the EMA as any medical occurrence that at any dose, results in death, is life-threatening, requires inpatient hospitalization or prolongs an existing hospitalization, results in persistent or significant disability/incapacity, is a congenital anomaly/birth defect, or is an important medical event that may jeopardize the patient or require intervention to prevent the aforementioned outcomes [31].No SAEs related to the study vaccines were reported.
talization, results in persistent or significant disability/incapacity, is a congenital anomaly/birth defect, or is an important medical event that may jeopardize the patient or require intervention to prevent the aforementioned outcomes [31].No SAEs related to the study vaccines were reported.
Subgroup analyses of neutralizing antibody responses against Omicron XBB.1.16and Omicron XBB.1.5among individuals aged 60 years or older, both with and without prior reported SARS-CoV-2 infections and subjects who received three or more prior doses, showed higher antibody titers with PHH-1V81 compared to BNT162b2 XBB.1.5.Safety endpoint frequencies were similar between the two booster groups, but PHH-1V81 demonstrated an overall lower reactogenicity profile, with a significantly higher proportion of subjects reporting no AE compared to BNT162b2 XBB.1.5.This aligns with the previously reported favorable safety profile of PHH-1V compared to BNT162b2 [18].It reinforces the notion that adjuvanted protein subunit vaccines, like PHH-1V81, are well-suited for vulnerable populations, including immunocompromised individuals, due to their safety profile and their ability to generate high levels of neutralizing antibodies, surpassing those induced by inactivated virus vaccines [32].
Other concerning, very rare AEs (<1 in 10,000), such as myocarditis reported with mRNA vaccine platforms, adjuvanted protein-based vaccines, and adenovirus vectorbased vaccines, have not been observed in previous studies with the PHH-1V prototype vaccine [33,34].According to currently available evidence, most cases of vaccine-associated myocarditis are mild, transient, and self-limiting.However, these infrequent AEs can have a worse prognosis in young males after two doses of mRNA vaccines, making them a more vulnerable population [35,36].Innate immunity, cytokines, and the inflammatory reaction may all play a crucial role [33].The PHH-1V and adapted PHH-1V81 vaccines have shown low reactogenicity, with no cases documented in previous studies [18,19,25,37].Similarly, a low incidence of myocardial events has also been observed with other protein-based vaccines [33].In this study, the adapted PHH-1V81 booster has been less reactogenic than the adapted BNT162b2 XBB.1.5booster.The low reactogenicity of PHH-1V and the adapted PHH-1V81, coupled with the low incidence of myocardial events documented with protein-based vaccines, has not justified the measurement of troponin levels, other biomarkers, or other inflammatory parameters in this clinical trial.Pharmacovigilance registries will likely provide sufficient data to better characterize the epidemiology of these rare AEs and their association with COVID-19 immunization [33].
The immunogenicity of PHH-1V81 against Omicron's XBB.1.16and XBB.1.5variants holds significant epidemiological importance amid Omicron's emergence as the predominant SARS-CoV-2 variant globally [38].The ongoing evolution of SARS-CoV-2 and the emergence of new variants, exemplified by the JN.1 variant, pose challenges with waning or inadequate protection from previous infections and vaccines, potentially allowing for the evasion of immunity and enhancing transmission [6,39].This is compounded by a population largely exposed to multiple variants through infection, vaccination, and boosting [40,41].Consequently, future vaccines may encounter challenges related to factors such as immune imprinting, immune seniority [42], or an immunoglobulin G (IgG) class switch [43].
In this complex scenario, it can be hypothesized that the squalene adjuvanted RBDbased vaccine can stimulate the innate immune system [44,45], and additionally, the RBD immune-focused approach can induce a better response against new and conserved epitopes in emerging variants [46,47], overcoming or minimizing the potential negative effects associated with previous exposures.Furthermore, the IgG4 class switch associated with repeated vaccination with mRNA vaccines suggests the potential for alternative platforms for subsequent immunizations [43,48].The results support these assumptions, as better responses were observed with PHH-1V81 compared to BNT162b2 XBB.1.5against XBB and JN.1.The data presented herein, 14 days post-booster, do not include the IgG subclass analysis, as it is planned to be conducted at trial termination.This will include longer time points and provide better insights of the IgG4 class switch, which is mostly reported at later time points [41,46].
These interim results align with earlier findings of broad immune responses against previously circulating variants of concern, including Wuhan-Hu-1, Beta, Delta, and Omicron BA.1 observed with PHH-1V (Bimervax ® ; HIPRA) [18,37].They underscore the value of incorporating alternative vaccine platforms and adjuvants to address the diminishing returns observed with successive mRNA vaccinations, ensuring sustained immunogenicity against evolving variants of the virus [49,50].In this interim analysis, established laboratory techniques, including PBNA, VNA, and ELISpot, were employed to evaluate immune responses.However, limitations stem from the interim nature of the results, the short follow-up period (14 days post-booster), limited statistical power for subgroup analyses, and a restricted representation of individuals aged 60 and older.Long-term observations of the effects of the PPH-1V81 booster will be conducted at trial termination, including assessments of IgG subclasses, particularly IgG4 levels, which peak around six months post-vaccination [43,48,50,51].

Conclusions
The interim results endorse PHH-1V81 as a promising booster, offering balanced immunogenicity and tolerability.This updated vaccine is key for addressing SARS-CoV-2 variability and population immunity, enhancing protection against current and emerging strains.Future research should prioritize a prolonged follow-up to confirm immune response persistence and effectiveness, especially among vulnerable groups.With a favorable safety profile and robust response against tested variants, including predominant JN.1, the adjuvanted RBD SARS-CoV-2 vaccine emerges as a compelling candidate for future COVID-19 vaccination strategies.

Figure 2 .
Figure 2. The humoral response against SARS-CoV-2 variants Omicron XBB.1.16,Omicron XBB.1.5,and Omicron JN.1 induced by BNT62n2 XBB and PHH-1V81 at day 14.(A) The GMT for the adjusted treatment against the Omicron XBB.1.16variant for each vaccinated group (PHH-1V81 group; n = 406 and BNT62b2 XBB; n = 193) by PBNA; (B) the GMT for the adjusted treatment against the Omicron XBB.1.5variant for each vaccinated group (PHH-1V81 group; n = 406 and BNT62b2 XBB; n = 193) by PBNA; (C) the GMT for the adjusted treatment against the Omicron JN.1 variant for a subset of n = 100 participants (n = 65 with PHH-1V81 and n = 35 with BNT62b2 XBB1.5) by VNA.Graphics A-C represent the mean GMT with 95% CI at baseline (with a bar) and 14 days after booster (dark grey bar); the upper numbers represent the mean GMFR at day 14 from the baseline.CI: confidence interval; GMT: geometric mean titer; GMFR: geometric mean fold rise.

Figure 2 .
Figure 2. The humoral response against SARS-CoV-2 variants Omicron XBB.1.16,Omicron XBB.1.5,and Omicron JN.1 induced by BNT62n2 XBB and PHH-1V81 at day 14.(A) The GMT for the adjusted treatment against the Omicron XBB.1.16variant for each vaccinated group (PHH-1V81 group; n = 406 and BNT62b2 XBB; n = 193) by PBNA; (B) the GMT for the adjusted treatment against the Omicron XBB.1.5variant for each vaccinated group (PHH-1V81 group; n = 406 and BNT62b2 XBB; n = 193) by PBNA; (C) the GMT for the adjusted treatment against the Omicron JN.1 variant for a subset of n = 100 participants (n = 65 with PHH-1V81 and n = 35 with BNT62b2 XBB1.5) by VNA.Graphics(A-C) represent the mean GMT with 95% CI at baseline (with a bar) and 14 days after booster (dark grey bar); the upper numbers represent the mean GMFR at day 14 from the baseline.CI: confidence interval; GMT: geometric mean titer; GMFR: geometric mean fold rise.These increases in neutralizing antibodies are reflected in a GMFR (95% CI) for adjusted treatment at day 14 post-booster vaccination: 12.76 (11.01, 14.78) for PHH-1V81 and 9.42

Figure 8 .
Figure 8.The percentage of subjects with solicited local and systemic adverse events through day 7 by vaccine arm.Solicited local adverse events and solicited systemic adverse events were reported MedDRA.PT from day 0 through day 7 for the safety population with the available data at the cutoff date.Data are shown as the percentage of subjects in relation to the safety population (n = 607; n = 409 in PHH-1V81 arm and n = 198 in BNT162b2 XBB.1.5arm).If a subject experienced more than

Figure 8 .
Figure 8.The percentage of subjects with solicited local and systemic adverse events through day 7 by vaccine arm.Solicited local adverse events and solicited systemic adverse events were reported MedDRA.PT from day 0 through day 7 for the safety population with the available data at the cut-off date.Data are shown as the percentage of subjects in relation to the safety population (n = 607; n = 409 in PHH-1V81 arm and n = 198 in BNT162b2 XBB.1.5arm).If a subject experienced more than one event, the subject is counted once for each type of event.PTs are ordered in the decreasing frequency of the total number of subjects with each adverse event in the PHH-1V81 group.n, the number of subjects in the population; PT, preferred term.

Table 1 .
The baseline demographics of the safety population by vaccine arm.
N: the number of subjects in the population; n: the number of subjects meeting the criterion, SD: Standard deviation.Vaccines 2024, 12, 840 7 of 17